WO2021181399A1 - Cellules stromales mésenchymateuses et vésicules extracellulaires pour le traitement d'infections virales, d'inflammation et de fibrose tissulaire - Google Patents

Cellules stromales mésenchymateuses et vésicules extracellulaires pour le traitement d'infections virales, d'inflammation et de fibrose tissulaire Download PDF

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WO2021181399A1
WO2021181399A1 PCT/IL2021/050274 IL2021050274W WO2021181399A1 WO 2021181399 A1 WO2021181399 A1 WO 2021181399A1 IL 2021050274 W IL2021050274 W IL 2021050274W WO 2021181399 A1 WO2021181399 A1 WO 2021181399A1
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mir
msc
protein
viral
virus
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PCT/IL2021/050274
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Aharon Brodie
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Exostem Biotec Ltd.
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Priority to US17/911,056 priority Critical patent/US20230105667A1/en
Priority to PCT/IL2021/050466 priority patent/WO2021214778A1/fr
Priority to US17/920,858 priority patent/US20230183809A1/en
Publication of WO2021181399A1 publication Critical patent/WO2021181399A1/fr

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Definitions

  • the present invention relates to mesenchymal stromal cells and extracellular vesicles and their uses for treating viral infections, inflammation, and tissue fibrosis.
  • MSCs Mesenchymal stromal cells
  • MSCs are mesoderm-derived cells that can be obtained from autologous or allogeneic sources. These cells can be derived from different types of stromal cells, including bone marrow, adipose tissues and dental pulp, or from the placenta and umbilical cord. MSCs are immune privileged, expressing low levels of MHC-I and in most cases lacking expression of MHC-II.
  • MSCs from different sources have been widely explored for the treatment of various inflammatory and degenerative disorders and demonstrated a high safety profile. Approximately 1,000 clinical trials using human MSCs are listed in clinicaltrials.gov, out of which more than 40 have reached phase III.
  • MSCs have the potential to differentiate into adipogenic, chondrogenic and osteogenic lineages, in addition to some degree of myogenic differentiation upon engraftment in muscle tissue. They home to sites of inflammation and injury and have low tumorigenic potential. They were also shown to augment the activity of endogenous cells and downregulate inflammation and fibrosis.
  • MSCs Many of MSCs effects are mediated by a large variety of secreted factors, such as cytokines, chemokines and growth factors.
  • MSCs secrete many extracellular vesicles (EVs), which can also mediate the various effects of cells by playing important roles in intercellular communication.
  • EVs extracellular vesicles
  • MSCs attenuate tissue damage, inhibit fibrotic remodeling and apoptosis, promote angiogenesis, stimulate endogenous stem cell recruitment and proliferation and reduce immune responses.
  • EVs are membrane-bound nanovesicles with diameters of 30-150 nm that contain multiple proteins, nucleic acid, lipids and other molecules in a tissue- and cell-specific manner. EVs are secreted by a large variety of cells. They play major roles in cell-cell interactions and in multiple physiological and pathological conditions. EVs have been demonstrated to exert therapeutic effects in various pathological conditions via the delivery of a diverse cargo including miRNAs, IncRNAs, DNA molecules, proteins and lipids. Interestingly, similar to MSCs, EVs can home to injured, inflamed or tumor sites. In addition, EVs can internalize into various cell types and deliver both endogenous and exogenous cargos. These characteristics provide the basis for the use of EVs as drug delivery vehicles.
  • EVs In addition to their therapeutic and drug delivery functions, EVs also serve as circulating biomarkers for various diseases and as mediators of disease pathogenesis. Thus, tumors or virally infected cells secrete large quantities of EVs. These are a rich source of molecules that reflect the activity of the originating cells. Due to their selective molecular packaging and stability against degrading enzymes, secreted EVs represent a viable and consistent reservoir of biomarkers in human fluids. Therefore, they emerge as attractive candidates for liquid biopsy.
  • MiRNAs are a family of highly conserved, small noncoding RNAs of approximately 22 nucleotides that inhibit gene expression by binding to the 3’ untranslated region (3’ UTR) of specific target mRNAs thereby inducing gene silencing. They play major roles in the regulation of various aspects of various disease development and progression.
  • Another group of non-coding RNAs that can be used in liquid biopsy are long non-coding RNAs (IncRNAs), a class of transcripts longer than 200 nucleotides that have limited protein coding potential.
  • IncRNAs can affect tumor growth by regulating associated gene expressions at the transcriptional, post- transcriptional or epigenetic levels and their aberrant expression has been reported as a potential measure for diagnosis and prognosis of different diseases.
  • SARS-CoV-2 a beta coronavirus
  • SARS-CoV-2 a beta coronavirus
  • ARDS Acute respiratory distress syndrome
  • SARS-CoV-2 SARS-CoV-2
  • macrophages One of the important components of the immune system that contribute to the hyperactivation of the cytokine storm.
  • Lung fibrosis is considered a complication of ARDS.
  • other organs such as cardiac and kidney have been also reported to be affected by cytokine storm and severe hyperinflammatory responses and in chronic pathological conditions.
  • the present invention provides mesenchymal stromal cells (MSCs) and extracellular vesicles comprising exogenous proteins.
  • Pharmaceutical compositions comprising MSCs and extracellular vesicles are also provided.
  • the present invention further provides a method of treating, preventing or ameliorating a viral infection.
  • a mesenchymal stem cell comprising an exogenous membrane associated peptide comprising a fragment of a viral peptide or virus - interacting protein.
  • an extracellular vesicle comprising an exogenous membrane associated peptide comprising a fragment of a viral peptide or virus - interacting protein.
  • At least one miR or anti-miR selected from the group consisting of anti-miR-214, anti- miR-214, anti-miR-21, anti-miR-199, anti-miR-130, anti-miR-31, anti-miR-103, anti- miR- 144, anti-miR- 1825, miR-30d, miR-140p, miR-532 or miR-190;
  • the virus interacting protein is a virus binding receptor.
  • the MSC is an umbilical cord (UC) MSC or a chorionic placenta (CH) MSC.
  • UC umbilical cord
  • CH chorionic placenta
  • the membrane associated peptide is a membrane embedded peptide comprising an extracellular domain of the virus interacting protein.
  • the fragment of a virus binding receptor is capable of binding the virus.
  • the virus binding receptor is a receptor employed for viral entry.
  • the virus is a coronavirus and the virus binding receptor is angiotensin-converting enzyme 2 (ACE2).
  • ACE2 angiotensin-converting enzyme 2
  • the ACE2 is a cleavage resistant ACE2 mutant.
  • ACE2 comprises the amino acid sequence of SEQ ID NO: 1 and wherein the mutant ACE2 comprises mutation of arginine 273 of SEQ ID NO: 1 to alanine.
  • the coronavirus is SARS-CoV-2.
  • the exogenous membrane associated protein comprises an extracellular fragment of a viral protein priming protein.
  • the viral protein is a virus spike protein.
  • the fragment of a viral protein priming protein is capable of priming the viral protein.
  • the receptor and the priming protein bind and prime the virus.
  • the viral protein priming protein is transmembrane protease, serine 2 (TMPRSS2).
  • the viral protein priming protein is an inactive mutant of the viral protein priming protein.
  • the viral peptide is a virus spike protein
  • the fragment comprises a receptor-binding domain (RBD) of the spike protein.
  • the RBD comprises amino acids 437-509 of SEQ ID NO: 2.
  • the MSC or EV further comprises an exogenous microRNA (miR), anti-miR, small interfering RNA (siRNA), antisense oligonucleotide (ASO) or mRNA that inhibits virus replication, inhibits inflammation, inhibits fibrosis, inhibits expression of a viral protein priming protein or a combination thereof.
  • miR microRNA
  • siRNA small interfering RNA
  • ASO antisense oligonucleotide
  • mRNA that inhibits virus replication, inhibits inflammation, inhibits fibrosis, inhibits expression of a viral protein priming protein or a combination thereof.
  • the viral protein priming protein is TMPRSS2 and the miR that inhibits expression of a viral protein priming protein is selected from miR-98-5p, let-7, and miR-4458.
  • the miR or anti-miR that inhibits inflammation is selected from: miR-124, miR-145, miR-20, miR-9, miR-506, miR-455, miR-27a, miR-29c, miR- 328, miR- 190, miR-532, and anti-miR-214.
  • the miR or anti-miR that inhibits inflammation is selected from: miR-124, miR-145, miR-29c, miR-328, miR- 190, miR-532, and anti-miR-214.
  • the miR or anti-miR that inhibits inflammation is selected from: miR-29c, miR-328, miR-190, miR-532, and anti-miR-214.
  • the MSC or EV comprise exogenous miR-124 and anti- miR-214.
  • the MSC or EV further comprises an anti-viral agent.
  • the antiviral agent is selected from a vaccine, a TMPRSS2 inhibitor, an ACE2 blocking agent, soluble ACE2, and an anti-inflammatory compound.
  • the anti-viral agent is selected from metformin and a cannabinoid.
  • the cannabinoid is cannabidiol (CBD).
  • the MSC or EV comprises at least two different membrane associated peptides comprising a fragment of a virus interacting protein.
  • the extracellular vesicle is from a plant cell, a bacterial cell, an animal cell or from milk.
  • the animal cell is an MSC.
  • the MSC is an MSCs of the invention.
  • the MSC or EV comprises at least one miR or anti -mi R selected from the group consisting of miR-29c, anti-miR-214, miR-190 and miR-328.
  • the MSC or EV has an anti-fibrotic effect in the lung.
  • the MSC or EV comprises at least one miR or anti -miR selected from the group consisting of anti-miR-214, miR-190 and miR-532.
  • the MSC or EV has an anti-fibrotic effect in the kidneys.
  • the MSC or EV comprises at least one molecule selected from the group consisting of miR-29, anti-miR-21, anti-miR-214 and/or Eluforsen.
  • the MSC or EV is for use in treating cystic fibrosis.
  • a pharmaceutical composition comprising any one of: a. an MSC of the invention, b. an extracellular vesicle of the invention, and c. a combination thereof.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or adjuvant.
  • a method of treating, preventing or ameliorating a viral infection in a subject in need thereof comprising administering to the subject at least one of: a. a pharmaceutical composition of the invention; b. a pharmaceutical composition comprising an MSC; c. a pharmaceutical composition comprising an extracellular vesicle from an MSC; and d. a pharmaceutical composition comprising isolated and purified extracellular vesicles; thereby treating, preventing or ameliorating the viral infection.
  • the MSC is selected from a UC MSC, and a CH MSC.
  • the viral infection is an infection by a virus bound by the virus binding protein.
  • the administering is systemic administration or administration to a site of infection.
  • the administration is selected from inhalation, intravenous, intranasal, intrathecal, intramuscular or oral administration.
  • the MSC is allogenic or autologous to the subject.
  • the method comprises administering a pharmaceutical composition comprising an MSC or EV of the invention.
  • the method further comprises administering an anti viral medication.
  • the anti-viral medication is selected from a cannabinoid and metformin.
  • the cannabinoid is cannabidiol (CBD).
  • Figure 1 A bar graph of percent decrease in fluorescence in lung cells incubated with fluorescent pseudovirus particles in the presence of EVs from unmodified MSC or MSCs expressing ACE2 protein.
  • Figure 4 A bar graph of the percent of cells that are fluorescent after additions of fluorescently labeled EVs.
  • Figures 6A-C Bar graphs of relative expression of (6A-B) TNFa and (6A, 6C) IL-6 in HBE cells incubated with recombinant SARS-CoV-2 peptides in the presence of (6A) MSC EVs, (6C) MSC EVs expressing miR mimics or (6B) in transwell culture with MSCs.
  • Figures 8A-C Bar graphs of relative TNFa expression in (8A) HBE cells and (8B-C) microglial cells treated with recombinant SARS-CoV-2 SI peptide in the presence of CH-MSCs (8B) with or without the combination of an additional anti-viral agents or (8C) with the antiviral agent loaded into the EVs.
  • Figures 9A-B Bar graphs of relative TGFP expression in (9A) kidney fibroblasts treated with recombinant SARS-CoV-2 S 1 peptide in the presence or absence of unmodified EVs or EVs loaded with miR-532, miR-190 or an anti-miR-214 as well as unmodified CH-MSCs or CH- MSCs expressing miR-190 and (9B) lung fibroblasts treated with recombinant SARS-CoV-2 SI peptide in the presence or absence of EVs loaded with miR-29c, miR-328 or an anti-miR-214.
  • Figure 10 Bar graph of relative luciferase expression from reporter cells treated with EVs from MSCs or milk expressing various miRs.
  • the present invention in some embodiments, provides for mesenchymal stromal cells (MSCs) and extracellular vesicles (EV) comprising any one of (i) an exogenous membrane embedded protein, (ii) a specific oligonucleotide repertoire (e.g., miR, anti-miR, antisense oligonucleotide); and combination thereof.
  • MSCs mesenchymal stromal cells
  • EV extracellular vesicles
  • the present invention further concerns pharmaceutical compositions comprising these MSCs, extracellular vesicles or a combination thereof. Methods of treating, preventing or ameliorating viral infections are also provided.
  • an MSC comprising an exogenous membrane associated peptide, wherein said exogenous membrane associated peptide comprises an extracellular fragment of a viral protein or virus interacting protein.
  • an extracellular vesicle comprising an exogenous membrane associated peptide, wherein said exogenous membrane associated peptide comprises an extracellular fragment of a viral protein or virus interacting protein.
  • an MSC or an EV derived therefrom comprising at least one subset of molecules selected from:
  • MSC are present in the bone marrow, adipose tissue, peripheral blood, chorionic placenta (CH), amniotic placenta, umbilical cord (UC) blood, and dental pulp, among other tissues.
  • the term "multipotent" refers to stem cells which are capable of giving rise to many cell types.
  • the MSC is derived from umbilical cord or chorionic placenta.
  • the MSC is derived from dental pulp, umbilical cord or chorionic placenta.
  • the MSC is derived from chorionic placenta.
  • the MSC is derived from umbilical cord.
  • the MSC is derived from dental pulp.
  • the MSC is derived from any one of umbilical cord and chorionic placenta. In some embodiments, the MSC is not derived from amniotic placenta. In some embodiments, MSC is derived from placenta. In some embodiments, the MSC is derived from amniotic placenta. In some embodiments, the MSC is derived from bone marrow. In some embodiments, the MSC is derived from adipose tissue. In some embodiments, the MSC is derived from umbilical cord blood. In some embodiments, the MSC is derived from peripheral blood. In some embodiments, the pharmaceutical composition is devoid of amniotic placenta MSCs.
  • the pharmaceutical composition is substantially devoid of amniotic placenta MSCs.
  • the MSC is not an adipose derived MSC.
  • the MSC is an unmodified MSC.
  • the MSC has been modified to over express at least one exogenous molecule.
  • the MSC is derived from a stem cell. In some embodiments, the MSC is differentiated from a stem cell. In some embodiments, the stem cell is a naturally occurring stem cell. In some embodiments, the stem cell is a human stem cell. In some embodiments, the stem cell is an adult stem cell. In some embodiments, the stem cell is an embryonic stem cell. In some embodiments, the stem cell is not an embryonic stem cell. In some embodiments, the stem cell is an umbilical cord stem cell. In some embodiments, the stem cell is a placental stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC). In some embodiments, the stem cell is a non-naturally occurring stem cell.
  • iPSC induced pluripotent stem cell
  • the MSC is derived from an iPSC. In some embodiments, MSC is differentiated from an iPSC. [083] In some embodiments, the MSC and/or its exosomes/extracellular vesicles are allogenic to the subject. In some embodiments, the MSC and/or its exosomes are autologous to the subject. In some embodiments, the MSC and/or its exosomes are allogenic or autologous to the subject. In some embodiments, the MSC and/or its exosomes do not induce an immune response in the subject. MSC and especially their exosomes and extracellular vesicles have a strong advantage as a therapeutic as they do not express MHCII molecules and do not induce an immune response.
  • MSCs and their exosomes actively inhibit the immune response.
  • CH and UC MSCs and their exosomes are particularly effective in this respect. In this way the MSCs and/or their exosomes can be used as an “off the shelf’ therapeutic agent that can be administered to any subject in need thereof.
  • Chorionic (CH), and umbilical cord (UC) MSCs are well known in the art.
  • these MSCs or their secreted vesicles can be identified by examining the expression of various proteins, and regulatory RNA such as are described in international patent application WO/2018083700, the content of which are herein incorporated by reference in their entirety.
  • the MSCs are identified by the tissue they were isolated from.
  • the MSC is an umbilical cord (UC) MSC. In some embodiments, the MSC is a chorionic placenta (CH) MSC.
  • UC umbilical cord
  • CH chorionic placenta
  • membrane associated is membrane embedded.
  • a membrane embedded peptide comprises a membranal domain.
  • a membranal domain is a transmembrane domain.
  • membrane associated is membrane anchored.
  • membrane anchored is lipid anchored.
  • the peptide is anchor via an anchoring peptide.
  • anchoring peptide is a glycosylphosphatidylinositol-linked protein (GPI).
  • a transmembrane domain is a membranal domain of a virus interacting protein.
  • a transmembrane domain is a transmembrane domain of a virus binding receptor.
  • the membranal domain is the native membranal domain of the protein comprising the fragment.
  • the fragment and membrane domain are from different proteins.
  • the exogenous membrane associated peptide comprise a fragment of a virus-interacting protein.
  • the fragment is an extracellular fragment.
  • the fragment is a capable of interacting with the virus.
  • the fragment is an extracellular domain.
  • the fragment is the full extracellular domain.
  • the fragment is the extracellular domain and membranal domain.
  • the fragment is the full protein.
  • interacting is binding.
  • the fragment of a virus binding receptor is capable of binding the virus.
  • the virus binding receptor is a receptor used for viral entry.
  • the virus interacting protein is employed for viral entry.
  • the virus interacting protein is a host cell surface component recognized by the virus. In some embodiments, recognized by the virus is recognized as a gateway of entry into the cell.
  • the virus is a coronavirus.
  • the virus binding receptor is angiotensin-converting enzyme 2 (ACE2).
  • the virus is a coronavirus, and the virus binding receptor is angiotensin-converting enzyme 2 (ACE2).
  • the virus interacting protein is transmembrane protease, serine 2 (TMPRSS2).
  • the virus interacting protein is transmembrane protease, serine 1 (TMPRSS1).
  • the ACE2 is a mutant ACE2.
  • the mutant ACE2 is a cleavage resistant ACE2 mutant.
  • ACE2 is human ACE2.
  • human ACE2 comprises the amino acid sequence: MS S S S WLLLSL V A VT A AQSTIEEQ AKTFLD KFNHE AEDLF Y QS SL AS WN YNTNITEEN V QNMNNAGDKWSAFFKEQSTFAQMYPFQEIQNFTVKFQFQAFQQNGSSVFSEDKSKRF NTIFNTMSTIYSTGKVCNPDNPQECFFFEPGFNEIMANSFDYNERFWAWESWRSEVGKQ FRPFYEEYVVFKNEMARANHYEDY GDYWRGDYEVNGVDGYDY SRGQFIEDVEHTFEE IKPFYEHFHAYVRAKFMNAYPSYISPIGCFPAHFFGDMWGRFWTNFYSFTVPFGQKPNI DVTDAMVDQAWDAQRIFKEAEKFFVSVGFPNMTQGFWENSMFTDPGNVQKAVCHPT AWDFGKGDFRIFMCTKVTMDDFFTAHHEMGHIQYDMAYAAQ
  • the cleavage resistant ACE2 comprises mutations of a plurality of lysine and arginine residues between amino acids 697 and 716 of SEQ ID NO: 1.
  • the mutant comprises at least 1, 2, 3, 4, 5 or 6 mutations. Each possibility represents a separate embodiment of the invention.
  • all lysines and arginines between amino acids 697 and 716 of SEQ ID NO: 1 are mutated.
  • the mutation is to a non-charged amino acid.
  • the mutation is to a non-polar amino acid.
  • the mutation is to a negatively charged amino acid.
  • the mutation is to alanine.
  • the cleavage resistant ACE2 comprises mutation of arginine 273 of SEQ ID NO: 1.
  • arginine 273 is mutated to alanine.
  • the coronavirus is an alpha coronavirus. In some embodiments, the coronavirus is 229E or NL63. In some embodiments, the coronavirus is a beta coronavirus. In some embodiments the coronavirus is OC43, HKU1 or MERS-CoV. In some embodiments, the coronavirus is SARS-CoV-1. In some embodiments, the coronavirus is SARS-CoV-2. In some embodiments, the coronavirus is selected from SARS-CoV-1 and SARS-CoV-2.
  • the MSC and/or extracellular vesicle comprises at least one exogenous membrane associated peptide comprising a fragment of a virus interacting protein. In some embodiments, the MSC and/or extracellular vesicle comprises a plurality of exogenous membrane associated peptide comprising a fragment of a virus interacting protein. In some embodiments, the MSC and/or extracellular vesicle comprises at least 1, 2, 3, 4, 5 or 6 different exogenous membrane associated peptide comprising a fragment of a virus interacting protein. In some embodiments, the MSC and/or extracellular vesicle comprises at least two different exogenous membrane associated peptide comprising a fragment of a virus interacting protein.
  • the exogenous membrane associated peptide comprises an extracellular fragment of a viral protein priming protein.
  • the viral protein is a virus spike protein.
  • the fragment of a viral protein priming protein is capable of priming a viral protein.
  • the receptor and the priming protein bind and prime the same virus.
  • the receptor and the priming protein both bind and prime a coronavirus.
  • the receptor and the priming protein both bind and prime SARS-CoV-1.
  • the receptor and the priming protein both bind and prime SARS-CoV-2.
  • the viral protein priming protein is transmembrane protease, serine 2 (TMPRSS2). In some embodiments, the viral protein priming protein is neuropilin-1 (NRRP1). In some embodiments, the priming protein is proteolytic. In some embodiments, the priming protein cleaves the receptor.
  • the viral protein priming protein is an inactive mutant of the viral protein priming protein.
  • inactive is inactive to prime.
  • in active is proteolytically inactive.
  • inactivating comprises the expression of an inactivating molecule.
  • the inactivating molecule is a peptide-conjugated morpholino.
  • the exogenous membrane associated protein comprises a fragment of a viral protein.
  • the viral protein is a viral surface protein.
  • the viral protein is a viral entry protein.
  • the viral protein is a spike protein.
  • SARS-CoV-2 spike protein (SI) comprises the amino acid sequence:
  • ECDIPIGAGICASYQTQTNSPRRAR SEQ ID NO: 2.
  • SARS-CoV-2 spike protein SEQ ID NO: 2.
  • the exogenous membrane associated protein comprises an extracellular fragment of a viral receptor binding domain (RBD).
  • the exogenous membrane associated protein comprises a viral RBD.
  • the fragment is the receptor binding motif of the RBD.
  • an MSC or EV comprising an RBD acts as a competitive inhibitor of the binding of the virus to its receptor.
  • an MSC or EV comprising an RBD is useful as a vaccine (e.g., generate antibodies towards the virus).
  • the exogenous membrane associated protein comprises the receptor binding domain (RBD) of SARS-CoV-1 and SARS-CoV-2 or any domain of the virus (e.g., in the S protein) that can inhibit the binding of the virus to ACE2 or an alternative receptor).
  • the exogenous membrane associated protein comprises the receptor binding domain (RBD) of SARS-CoV-1 or SARS-CoV-2.
  • RBD comprises amino acid residues 331-524 of SARS-CoV-2 S protein (e.g., SEQ ID NO:2) or amino acid residues 318-510 of SARS-CoV- 1 S protein.
  • the receptor binding motif of the RBD comprises amino acids 437-509 of SEQ ID NO: 2.
  • the receptor binding motif of the RBD consists of amino acids 437-509 of SEQ ID NO: 2. In some embodiments, the RBD comprises amino acids 319-541 of SEQ ID NO: 2. In some embodiments, the RBD consists of amino acids 319- 541 of SEQ ID NO: 2.
  • the RBD comprises RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTF KCY GVSPTKLNDLCFTNVY ADSFVIRGDEVRQIAPGQTGKIAD YNYKLPDDFTGCVIA WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQ SYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF (SEQ ID NO: 4).
  • the RBD consists of SEQ ID NO: 4.
  • an MSC and/or extracellular vesicle of the invention comprises an exogenous nucleic acid molecule that inhibits virus replication, inhibits inflammation, inhibits fibrosis, inhibits expression of a viral protein priming protein or a combination thereof.
  • the exogenous nucleic acid molecule inhibits virus replication.
  • the exogenous nucleic acid molecule inhibits inflammation.
  • the exogenous nucleic acid molecule inhibits fibrosis.
  • the exogenous nucleic acid molecule inhibits expression of a viral protein.
  • the exogenous nucleic acid molecule inhibits expression of a viral protein priming protein.
  • the exogenous nucleic acid molecule is selected from a microRNA (miR), an anti-miR, a small interfering RNA (siRNA), an antisense oligonucleotide (ASO) or an mRNA.
  • miR microRNA
  • siRNA small interfering RNA
  • ASO antisense oligonucleotide
  • the miR and/or an anti-miR are over-expressed in the MSC or EV derived therefrom.
  • the miR is a miR mimic. In some embodiments, the miR mimic is not a molecule found in nature. In some embodiments, the mimic is chemically modified. In some embodiments, the chemical modification extends the half-life of the mimic. In some embodiments, chemically modified is a chemically modified backbone. Chemical modification is well known in the art and includes, for example, 2-MOE, LNA, PNA, phosphorothioate and the like.
  • an anti-miR is an antagomir.
  • an anti-mir is a molecule not found in nature.
  • the anti-mir is chemically modified.
  • the ASO is a molecule not found in nature. In some embodiments, the ASO is chemically modified.
  • the antisense oligonucleotide (ASO), siRNA or both targets viral non-coding RNA (ncRNA).
  • ncRNA viral non-coding RNA
  • the ncRNA is from SARS-CoV-1.
  • the ncRNA is from SARS-CoV-2.
  • the ncRNA is nsp3.1.
  • the ncRNA is nsp3.2.
  • the siRNA binds to and inhibits an mRNA encoding TNF-alpha converting enzyme (TACE).
  • TACE TNF-alpha converting enzyme
  • the ASO binds to and inhibits an mRNA encoding TACE.
  • an “exogenous miR” refers to expression of a miR, miR mimic or other synthetic version of the miR that has been introduced into the cell.
  • the cell may express an endogenous form of the miR, but this refers to an externally introduced synthetic form of the miR.
  • synthetic is comprising chemical modification.
  • the miR inhibits expression of a viral protein priming protein.
  • the viral protein priming protein is TMPRSS2.
  • the miR that inhibits expression of a viral protein priming protein is selected from miR-98-5p, let-7, miR- 4458, miR-4500 and miR-582, and any combination thereof.
  • the miR that inhibits expression of a viral protein priming protein is selected from miR-98-5p, let-7, and miR- 4458.
  • the miR is selected from miR-98-5p, let-7, and miR-4458.
  • the miR is miR-98-5p.
  • the miR is let-7.
  • the miR is miR-4458.
  • the miR or anti-miR inhibits and/or reduces fibrosis and inflammation.
  • the anti-miR is anti-miR-214.
  • the anti-miR is anti -miR -21.
  • the miR inhibits and/or reduces inflammation.
  • the miR is selected from: miR-145, let-7, miR-1, miR-106, miR-103, miR-10, miR- 107, miR-1185, miR-124, miR-1226, miR-1271, miR-128, miR-129, miR-1303, miR-17, mir-93, miR-20, miR-106, miR- 155 and miR- 130, and any combination thereof.
  • the miR or anti-miR is selected from the group consisting of anti-miR-21, anti-miR-214, miR-124, miR-145 or miR-27a, miR-21, miR-155, miR-20, miR-9, miR-506, miR-124, and miR-455. In some embodiments, the miR or anti-miR is selected from the group consisting of anti-miR-214, miR-124, miR-145 or miR-27a, miR-21, miR-155, miR-20, miR-9, miR-506, miR-124, and miR- 455.
  • the miR or anti-miR is selected from the group consisting of anti-miR- 21, miR-124, miR-145 or miR-27a, miR-21, miR-155, miR-20, miR-9, miR-506, miR-124, and miR-455. In some embodiments, the miR or anti-miR is selected from the group consisting of miR- 29c, miR-328, miR- 190, miR-532 and anti-miR-214.
  • the miR or anti-miR is selected from the group consisting miR-124, miR-145, miR-20, miR-9, miR-506, miR-455, miR-27a, miR-29c, miR-328, miR-190, miR-532, and anti-miR-214. In some embodiments, the miR or anti-miR is selected from the group consisting miR-124, miR-145, miR-29c, miR-328, miR-190, miR-532, and anti-miR-214. In some embodiments, the miR or anti-miR is miR-124 and anti-miR-214.
  • the MSCs comprises miR-124 and anti-miR-214.
  • the EVs comprises miR-124 and anti-miR-214.
  • the miR is miR-29c.
  • the miR is miR-328.
  • the miR is miR-190.
  • the miR is miR-532.
  • the anti-miR is anti-miR-214.
  • the miR is miR-124.
  • the miR is miR-145.
  • the miR inhibits and/or reduces inflammation, such as in a pathological condition associated with a hyper pro-inflammatory response (e.g., ARDS, cytokine storm and the like).
  • a pathological condition associated with a hyper pro-inflammatory response e.g., ARDS, cytokine storm and the like.
  • the miR is selected from the group consisting of miR- 570-3p, miR-125a-5p, hsa-miR-124, hsa-miR-34, hsa-miR-132, hsa-miR-146a, hsa-miR-223, and hsa-let-7c.
  • the miR or anti-miR inhibits and/or reduces viral replication.
  • the miR or anti-miR that inhibits viral replication is selected from miR-5197- 3p, miR-511-3p, anti-miR-9 and miR-3914, and any combination thereof.
  • the miR or anti-miR that inhibits viral replication is selected from miR- 124 and miR- 145.
  • the miR is miR-124.
  • the miR is miR-145.
  • the one or more miRs inhibits and/or reduces fibrosis.
  • the miR is miR-145.
  • the miR is miR-145, miR-29c, anti- miR-214 and miR-328.
  • the miR is miR-145, miR-29c, anti -miR -21 and miR-328.
  • the miR is miR-145, miR-29c, anti-miR-214, anti-miR-21 and miR-328.
  • the one or more miRs or anti-miRs inhibits and/or reduces lung fibrosis and inflammation. In some embodiments, the one or more miRs or anti-miRs inhibits and/or reduces lung fibrosis. In some embodiments, the miR or anti-miRs is at least one miR or anti-miR selected from the group consisting of anti-miR-21, anti-miR- 181 , anti-miR-214, anti- miR-1246, anti-miR-199, or overexpression (OE) of miR 29c, 27a, 31, 124, 127 or 26a.
  • OE overexpression
  • the miR or anti-miRs is at least one miR or anti-miR selected from the group consisting of miR-29c, miR-328, and anti-miR-214. In some embodiments, the miR or anti-miRs is at least one miR or anti-miR selected from the group consisting of miR- 29c, miR-328, miR- 190 and anti-miR-214. In some embodiments, the miR is miR-29c. In some embodiments, the miR is miR-328. In some embodiments, the miR is miR- 190. In some embodiments, the anti-miR is anti- miR-214.
  • the one or more miRs or anti-miRs inhibits and/or reduces cystic fibrosis.
  • the miR or anti-miRs is at least one miR or anti-miR selected from the group consisting of miR-29, anti-miR-214 and/or Eluforsen.
  • the miR is at least one miR selected from the group consisting of miR-29, anti-miR-21 and/or Eluforsen.
  • Eluforsen refers to a 33 nt, single-stranded, fully phosphorothioated and fully 2 ’-O-methyl-modified oligonucleotide partly complementary to the p.Phe508del-CFTR RNA, and based on the CF4 molecule (5’- AUCAUAGGAAACACCAAAGAUGAUAUUUUCUUU-3 ’ ; SEQ ID NO: 3).
  • any Eluforsen includes any molecule having the same effect as Eluforsen.
  • the one or more oligonucleotide molecule inhibits and/or reduces liver fibrosis.
  • the oligonucleotide molecule is selected from miR-30a, miR- 9, miR-92-3p, a tribbles pseudokinase 3 (TRIB3) silencing molecule.
  • TRIB3 silencing molecule include microRNA (miR), an anti-miR, a small interfering RNA (siRNA), and an antisense oligonucleotide (ASO).
  • the one or more miRs or anti-miRs inhibit and/or reduces liver fibrosis.
  • the miR or anti-miR is at least one miR or anti-miR selected from the group consisting of anti-miR-214, anti-miR-21, anti-miR-199, anti-miR-130, anti-miR-31, anti-miR-103, anti-miR-144, anti-miR- 1825, miR-30d, miR-140p, miR-532 or miR-190.
  • the one or more miRs or anti-miRs inhibit and/or reduce kidney fibrosis.
  • the miR or anti-miR is at least one miR or anti-miR selected from miR-532, miR-190 and anti-miR-214.
  • the miR is miR-532.
  • the miR is miR-190.
  • the anti-miR is anti-miR-214.
  • the MSC of the invention or an extracellular vesicle of the invention are administered in combination with an anti-viral agent.
  • anti-viral agents include metformin, hydroxychloroquine, and melatonin.
  • an MSC of the invention or an extracellular vesicle of the invention further comprises an anti-viral agent.
  • a composition of the invention further comprises an anti-viral agent.
  • the antiviral agent is selected from a vaccine, a TMPRSS2 inhibitor, an ACE2 blocking agent, soluble ACE2, and an anti-inflammatory compound.
  • the anti-inflammatory compound is a cannabinoid.
  • the antiviral agent is a vaccine.
  • the antiviral agent is a TMPRSS2 inhibitor.
  • the antiviral agent is an ACE blocking agent.
  • the antiviral agent is a soluble ACE2.
  • the antiviral agent is an anti-inflammatory compound.
  • the virus interacting protein is fused to an Fc portion of an immunoglobulin.
  • the ACE2 is fused to an Fc portion of an immunoglobulin.
  • the ACE2 blocking agent is an anti-ACE2 antibody.
  • the antibody is a blocking antibody.
  • the antibody is non- cytotoxic.
  • the antibody is an immunoglobulin-G2 (IgG2) or IgG4 antibody.
  • the anti-viral agent is a cannabinoid.
  • the cannabinoid is selected from cannabidiol (CBD), tetrahydrocannabinol (THC) and a combination thereof.
  • the cannabinoid is CBD.
  • the CBD comprises at least one of: cannabidiol-C4 (CBD-C4), cannabidiol (CBD-C5), cannabidiol momomethyl ether, cannabidiolic acid (CBD-A), cannabigerolic acid (CBN-A), cannabigerol (CBG), cannabinol (CBN), cannabinolic acid (CBN- A), cannabichromenic acid (CBC-A), cannabichromene (CBC), cannabidivarin, cannabidiorcol, cannabidivarinic acid, CBD-V, THC-V and any combination thereof.
  • CBD-V cannabidiol-C4
  • CBD-C5 cannabidiol
  • CBD-V cannabidiol-C5
  • CBD-V cannabidiol-C5
  • CBD-V cannabidiol-C5
  • CBD-V cannabidiol-C5
  • CBD-V
  • the cannabinoid is selected from CBD, cannabidiol momomethyl ether, cannabidiolic acid (CBD-A), cannabigerolic acid (CBN-A), cannabigerol (CBG), cannabinol (CBN), cannabinolic acid (CBN- A), cannabichromenic acid (CBC-A), cannabichromene (CBC), cannabidivarin, cannabidiorcol, cannabidivarinic acid, CBD-V, THC-V and any combination thereof.
  • the CBD is selected from cannabidiol-C4 (CBD-C4), cannabidiol (CBD-C5).
  • the THC comprises at least one of: (-)-A 9 - tetrahydrocannabinol (THC), 2-carboxy-THC (THCA), an ester of THCA, 4-carboxy-THC (THCA-B), an ester of THCA-B, 11-OH-THCA, THC-l l-oic acid, A8-THC-11-oic acid, 1,1- dimethylheptyl-A8-THC-7-oic acid or any combination thereof.
  • the cannabinoid may be any isolate or purity of cannabinoid. In some embodiments, the cannabinoid is an essentially pure cannabinoid. In some embodiments, the cannabinoid is an isolated cannabinoid. In some embodiments, the cannabinoid is not psychoactive.
  • the anti-viral agent is metformin.
  • the anti-inflammatory compound is metformin.
  • the anti inflammatory compound is curcumin.
  • the present invention comprises an isolated extracellular vesicle of an MSCs of the invention.
  • the extracellular vesicle is an isolated extracellular vesicle.
  • the extracellular vesicle is a purified extracellular vesicle.
  • extracellular vesicle refers to membrane bound vesicles that can be divided into three main groups: exosomes, microvesicles and apoptotic bodies. Exosomes are small membrane vesicles of endocytic origin with a size of 50-100nm.
  • exosomes can be loaded with various drugs and exogenous nucleic acids or proteins and deliver this cargo to different cells.
  • extracellular vesicles and “exosomes” are the same and used interchangeably.
  • the extracellular vesicle is any natural or synthetic vesicle having similar functionality and/or activity as the extracellular vesicles of the invention. None- limiting examples of such vesicle include microvesicles, liposomes, micelles and the like.
  • the extracellular vesicles are cell-derived vesicles.
  • the extracellular vesicle is from a plant cell.
  • the extracellular vesicle is from a bacterial cell.
  • the extracellular vesicle is from an animal cell.
  • the extracellular vesicles are cell-derived vesicles secreted from MSCs.
  • the extracellular vesicles are milk derived vesicles.
  • the animal is a mammal.
  • the extracellular vesicles from a mammal are from milk.
  • the extracellular vesicle is from an animal cell.
  • the animal cell is an MSC.
  • the extracellular vesicle is from an animal MSC cell and the exogenous membrane associated protein is added directly to the extracellular vesicle.
  • Methods of adding exogenous molecules to vesicles are well known in the art and include but are not limited to mixing, passive diffusion, active diffusion, and membrane anchoring. Any such method may be used in practicing the invention.
  • membrane association is by charge interaction. In some embodiments, membrane association is not by charge interaction.
  • the present invention comprises a pharmaceutical composition comprising any one of: an MSC of the present invention, an extracellular vesicle of the present invention and a combination thereof.
  • the pharmaceutical composition comprises an MSC of the present invention.
  • the pharmaceutical composition comprises an extracellular vesicle of the present invention.
  • the pharmaceutical composition comprises a combination of a MSC of the present invention and an extracellular vesicle of the present invention.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient or adjuvant.
  • carrier refers to any component of a pharmaceutical composition that is not the active agent.
  • pharmaceutically acceptable carrier refers to non toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, formulation auxiliary of any type, or simply a sterile aqueous medium, such as saline.
  • sugars such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl
  • substances which can serve as a carrier herein include sugar, starch, cellulose and its derivatives, powered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer solutions, cocoa butter (suppository base), emulsifier as well as other non-toxic pharmaceutically compatible substances used in other pharmaceutical formulations.
  • Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, excipients, stabilizers, antioxidants, and preservatives may also be present.
  • any non-toxic, inert, and effective carrier may be used to formulate the compositions contemplated herein.
  • Suitable pharmaceutically acceptable carriers, excipients, and diluents in this regard are well known to those of skill in the art, such as those described in The Merck Index, Thirteenth Edition, Budavari et ah, Eds., Merck & Co., Inc., Rahway, N.J. (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the “Inactive Ingredient Guide,” U.S. Food and Drug Administration (FDA) Center for Dmg Evaluation and Research (CDER) Office of Management, the contents of all of which are hereby incorporated by reference in their entirety.
  • CTFA Cosmetic, Toiletry, and Fragrance Association
  • Examples of pharmaceutically acceptable excipients, carriers and diluents useful in the present compositions include distilled water, physiological saline, Ringer's solution, dextrose solution, Hank's solution, and DMSO. These additional inactive components, as well as effective formulations and administration procedures, are well known in the art and are described in standard textbooks, such as Goodman and Gillman’s: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al. Eds. Pergamon Press (1990); Remington’s Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa.
  • compositions may also be contained in artificially created structures such as liposomes, ISCOMS, slow- releasing particles, and other vehicles which increase the half-life of the peptides or polypeptides in serum.
  • liposomes include emulsions, foams, micelies, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like.
  • Liposomes for use with the presently described peptides are formed from standard vesicle-forming lipids which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol.
  • the selection of lipids is generally determined by considerations such as liposome size and stability in the blood. A variety of methods are available for preparing liposomes as reviewed, for example, by Coligan,
  • the carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.
  • the present invention comprises a method of treating, preventing or ameliorating a viral infection in a subject in need thereof, the method comprising administering to a subject at least one of: a. a pharmaceutical composition described herein; b. a pharmaceutical composition comprising an MSC; c. a pharmaceutical composition comprising an extracellular vesicle from an MSC; and d. a pharmaceutical composition comprising isolated and purified extracellular vesicles; thereby treating, preventing or ameliorating said viral infection.
  • a pharmaceutical composition described herein a pharmaceutical composition described herein; b. a pharmaceutical composition comprising an MSC; c. a pharmaceutical composition comprising an extracellular vesicle from an MSC; and d. a pharmaceutical composition comprising isolated and purified extracellular vesicles; for use in treating, preventing or ameliorating a viral infection in a subject in need thereof.
  • the present invention comprises a method of treating, preventing or ameliorating an inflammation in a subject in need thereof, the method comprising administering to a subject at least one of: a. a pharmaceutical composition described herein; b. a pharmaceutical composition comprising an MSC; c. a pharmaceutical composition comprising an extracellular vesicle from an MSC; and d. a pharmaceutical composition comprising isolated and purified extracellular vesicles; thereby treating, preventing or ameliorating said inflammation in the subject.
  • a pharmaceutical composition described herein a pharmaceutical composition described herein; b. a pharmaceutical composition comprising an MSC; c. a pharmaceutical composition comprising an extracellular vesicle from an MSC; and d. a pharmaceutical composition comprising isolated and purified extracellular vesicles; for use in treating, preventing or ameliorating inflammation in a subject in need thereof.
  • the inflammation is a result of an infection such as a virus including but not limited to SARS-CoV-1 or SARS-CoV-2.
  • the inflammation is SARS-CoV-2 induced inflammation.
  • the present invention comprises a method of treating, preventing or ameliorating fibrosis in a subject in need thereof, the method comprising administering to a subject at least one of: a. a pharmaceutical composition described herein; b. a pharmaceutical composition comprising an MSC; c. a pharmaceutical composition comprising an extracellular vesicle from an MSC; and d. a pharmaceutical composition comprising isolated and purified extracellular vesicles; thereby treating, preventing or ameliorating said fibrosis in the subject.
  • a pharmaceutical composition described herein a pharmaceutical composition described herein; b. a pharmaceutical composition comprising an MSC; c. a pharmaceutical composition comprising an extracellular vesicle from an MSC; and d. a pharmaceutical composition comprising isolated and purified extracellular vesicles; for use in treating, preventing or ameliorating fibrosis in a subject in need thereof.
  • the fibrosis (e.g., lung fibrosis) is a result of ARDS. In some embodiments, the fibrosis (e.g., lung, cardiac, kidney fibrosis) is a result of cytokine storm. In some embodiments, the fibrosis (e.g., lung, cardiac, kidney fibrosis) is a result of a severe hyperinflammatory responses (e.g., to a virus including but not limited to SARS-CoV-1 or SARS- CoV-2). In some embodiments, the fibrosis (e.g., lung, cardiac, kidney fibrosis) is a result of a chronic pathological condition. In some embodiments, the fibrosis is SARS-CoV-2 induced fibrosis.
  • the method comprises administering the pharmaceutical composition described herein. In some embodiments, the method comprises administering a pharmaceutical composition comprising an MSC. In some embodiments, the method comprises administering a pharmaceutical composition comprising an extracellular vesicle from an MSC. In some embodiments, the method comprises administering a pharmaceutical composition comprising isolated and purified extracellular vesicles. In some embodiments, the method comprises administering a pharmaceutical composition comprising a combination of these compositions. Each possibility represents a separate embodiment of the invention.
  • the MSC is a UC MSC. In some embodiments, the MSC is a CH MSC.
  • the MSC is an unmodified MSC.
  • the extracellular vesicle is an unmodified extracellular vesicle.
  • the pharmaceutical composition comprises isolated extracellular vesicles.
  • the pharmaceutical composition comprises purified extracellular vesicles.
  • purified comprises a purity of at least 70, 75, 80, 85, 90, 95, 97, 99 or 100%. Each possibility represents a separate embodiment of the invention.
  • the pharmaceutical composition is devoid of cells.
  • the pharmaceutical composition is depleted of cells.
  • the pharmaceutical composition is devoid of non-MSC cells.
  • the pharmaceutical composition is depleted of non-MSC cells.
  • treatment encompasses alleviation of at least one symptom thereof, a reduction in the severity thereof, or inhibition of the progression thereof. Treatment need not mean that the disease, disorder, or condition is totally cured.
  • a useful composition herein needs only to reduce the severity of a disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide improvement to a patient or subject’s quality of life.
  • subject refers to an animal, more particularly to non-human mammals and human organism.
  • Non -human animal subjects may also include prenatal forms of animals, such as, e.g., embryos or fetuses.
  • Non-limiting examples of non-human animals include: horse, cow, camel, goat, sheep, dog, cat, non-human primate, mouse, rat, rabbit, hamster, guinea pig, pig.
  • the subject is a human.
  • Human subjects may also include fetuses.
  • a subject in need thereof is a subject afflicted with a fractured bone, a bone injury, diminished bone mass and/or bone abnormality.
  • the terms “subject” or “individual” or “animal” or “patient” or “mammal,” refers to any subject, particularly a mammalian subject, for whom therapy is desired, for example, a human.
  • a subject in need thereof is infected with a viral infection.
  • a subject in need thereof is infected with a coronavirus.
  • a subject in need thereof is infected with a coronavirus caused by SARS-CoV-1.
  • a subject in need thereof is infected with a coronavirus caused by SARS-CoV-2.
  • the viral infection is an infection by a virus bound by the virus binding protein.
  • One aspect of the present subject matter provides for oral administration of a therapeutically effective amount of a composition of the present subject matter to a patient in need thereof.
  • Other suitable routes of administration can include parenteral, subcutaneous, intravenous, intramuscular, anal, oral, intrathecal intranasal, or intraperitoneal.
  • the administration is intravenous.
  • the administration is intranasal.
  • the administration is intrathecal.
  • the administration is intramuscular.
  • the administration is oral.
  • the administration is by inhalation.
  • the administering is systemic administration or administration to a site of infection.
  • administration is systemic administration.
  • administration is administration to a site of infection.
  • administration is to the lungs.
  • the dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • the MSCs are allogenic to the subject. In some embodiments, the MSCs are autologous to the subject.
  • extracellular vesicles use of extracellular vesicles is superior to use of cells. It will be understood that as extracellular vesicles do not contain cellular machinery, they cannot facilitate viral replication. Thus, if the virus binds the vesicle this is no chance of the virus infecting an extracellular vesicle and reproducing as there would be with a cell. An extracellular vesicle can thus act as a decoy to bind virus without allowing spread of the infection.
  • a length of about 1000 nanometers (nm) refers to a length of 1000 nm+- 100 nm.
  • Example 1 EVs expressing ACE2 receptor block pseudovirus entry
  • pseudotyped SARS- Cov-2 viral particle was engineered.
  • Replication restricted recombinant pseudotyped lentiviral particles were generated containing the SI protein from SARS-CoV-2.
  • the viral particles also encoded a lucif erase reporter protein.
  • the viral particles were incubated with human lung epithelial cells expressing ACE2 and viral entry was monitored by measuring the luciferase activity of the cells. The relative increased luciferase activity of cells incubated with viral particles was a sign of viral entry.
  • MSCs from chorionic placenta (CH) or from umbilical cord (UC) were transfected with an expression vector encoding either full length WT ACE2 protein or an ACE2 variant, R273A. Transduction with a lentiviral vector was also performed. EVs were isolated from the culture media of these cells and in order to test their effects on virus entry neutralization, the EVs were preincubated with the pseudovirus particles for 1 hour before they were incubated with the lung cells. EVs from non-transfected MSCs were used as a negative control. As can be seen in Figure 1, all of the EVs containing ACE2 were highly effective at preventing virus entry, with a decrease in luciferase activity of at least 90%.
  • EVs were also generated expressing the full SARS-CoV-2 receptor-binding-domain (RBD) of the SARS-CoV-2 RBD protein or the S 1 peptide. These EVs were preincubated with the ACE2 expressing lung cells before the addition of the pseudovirus virus particles. These EVs protected the lung cells from infection in a comparable manner to the ACE2 expressing EVs.
  • Example 2 MSCs and/or EVs for treating ARDS, cytokine storm and tissue fibrosis
  • Acute respiratory distress syndrome is a lung injury caused by hyper pro- inflammatory response.
  • One of the factors that contribute to ARDS such as in subjects infected with SARS-CoV-2, is the hyperactivation of the immune system in response to virus infection.
  • One of the important components of the immune system that contribute to the hyperactivation of the cytokine storm is macrophages.
  • chorionic (CH) and umbilical cord (UC)- derived MSCs and extracellular vesicles (EVs) secreted by these cells, induce high levels of the IL-10 in LPS-treated macrophages and decreased the secretion of IL-6 and TNF-alpha that contribute to cytokine storm.
  • these two MSC subpopulations induced higher expression of VEGF and KGF.
  • MSCs and EVs derived from chorionic placenta exerted the strongest anti-inflammatory and pro-M2 effects followed by umbilical cord (Cord) derived MSCs and EVs and then by amniotic membrane (AM) derived MSCs and EVs.
  • Bone marrow (BM) and PL-MSCs (decidua basalis) derived cells also had an effect though it was greatly reduced, and adipose (AD) derived cells were the least effective.
  • the extracellular vesicles (EVs) of these cells had comparable effects, with CH and Cord EVs having the greatest effect.
  • EVs expressing the combination of miR- 124 and anti-miR-214 have the greatest potential in inhibiting pro-inflammatory response in LPS- activated THP-1 cells. These EVs inhibited the production of IL-6 by 81 % and that of TNF-alpha by 77.65%. Similarly, EVs carrying miR-145 or miR-27a and anti-miR-214 exerted a similar effect. In contrast, the combination of miR-155 and anti-miR-214 did not reduce the expression of TNF-alpha and IL-6 but rather increased their expression by 19 and 22 % respectively.
  • miRs inhibiting the NF-KB pathway include any one of miR-20, miR-9, miR-506, miR-124 or miR-455 in combination with miR-21 and miR-155 inhibit pro- inflammatory responses.
  • This Example shows the various inflammatory disorders associated with hyperactivation of the immune system, including but not limited to ARDS, sepsis, and cytokine storm, can be treated using subpopulations of MSCs and EVs.
  • Cord and placenta derived MSCs, specifically chorionic MSCs and/or their EVs may be administered locally, intravenously, intranasally, or by inhalation and decrease lung damage, inflammatory response, promote angiogenesis and regeneration.
  • Example 3 MSCs and/or EVs for treating COPD, pneumonia and emphysema
  • lung endothelial cells were either co-cultured with different MSC subpopulations in transwell plates (1 mM filters) or with purified EVs isolated from MSC cultures. The cells were then stimulated with LPS ( 1 mg/ml) for 24 hr. Cell death was determined using the live/dead assay. As presented in Figure 5, there was a differential protective effect of specific cells and EVs on the cells from LPS-induced cell death. CH-MSCs were so protective that they completely abrogated the effect of the LPS, and indeed cell death was reduced even as compared to the untreated control.
  • CH-MSC EVs were superior to all other cell types tested and all other EVs but were not as extraordinarily protective as the CH-MSCs themselves.
  • Example 4 MSCs and/or EVs for inhibiting SARS-CoV-2 induced inflammation
  • HBE cells Immortalized human bronchial epithelial (HBE) cells were incubated with 100 ng/ml His- SARS-CoV-2 spike recombinant SI subunit (SI) or His-SARS-Cov-2-RBD (RBD) for 24 hr. Inflammation was monitored by measuring the expression of TNF alpha and IL-6. Both recombinant proteins produced an inflammatory effect with TNFa and IL-6 both increasing by 4- 5 fold (Fig. 6A). EVs from unmodified CH-MSCs and UC-MSCs had a mild effect, reducing relative expression by -20-25%.
  • SI SI subunit
  • RBD His-SARS-Cov-2-RBD
  • EVs from MSC that were made to express either WT ACE2 or variant ACE2 were preincubated (1 hour) with the recombinant proteins before they were administered to the HBE cells. This produced a highly significant reduction in expression of the inflammatory markers with the levels returning nearly to normal (Fig. 6A). This indicates that the EVs were able to compete with the SI protein’s binding and activation of cellular ACE2. Similar results were observed regardless of whether WT or variant ACE2 was used.
  • EVs expressing RBD were preincubated with the HBE cells before addition of the RBD recombinant protein. These EVs were also found to be highly effective at reducing inflammatory expression, as for example TNFa levels were reduced by 50% when EVs from CH- MSCs expression RBD were used.
  • Example 5 MSCs and/or EVs for inhibiting SARS-CoV-2 induced microglial activation
  • Immortalized human microglia cells (overexpressing ACE2) were incubated with 100 ng/ml His-SARS-CoV-2 spike recombinant SI subunit (SI) for 24 hr.
  • SI SI subunit
  • the expression of TNFa was determined by RT-PCR as a marker for microglial activation.
  • the recombinant protein did activate the microglia producing a greater 3.5-fold increase (Fig. 7).
  • EVs from MSC that were made to express WT ACE2 were preincubated ( 1 hour) with the recombinant proteins before they were administered to the microglial cells. This produced a highly significant reduction in expression of TNFa of -50% (Fig. 7). This indicates that the EVs were able to compete with the SI protein’s binding and activation of microglial ACE2.
  • Example 6 Anti-viral combined therapy using MSCs and/or EVs
  • MSCs and EVs can be combined with different drugs (metformin, hydroxychloroquine, melatonin and other anti-viral drugs) to produce a more robust anti-viral effect.
  • HBE cells were incubated with SARS-Cov-2 SI peptide as before, and TNFa expression was quantified as a marker for inflammation.
  • CH-EVs produced a modest reduction in inflammation as before, as did two different anti-viral treatments (CBD and metformin) when administered alone.
  • CBD and metformin anti-viral treatments
  • the combination of the EVs with each treatment produced a greater (although additive) response (Fig. 8A). Similar results were observed with immortalized human microglial cells overexpressing ACE2 (Fig. 8B).
  • EVs from CH-MSCs were electroporated with either GFP alone or GFP and CBD. Fluorescence was monitored and it was determined that EVs were successfully loaded. Following electroporation, the EVs were added to immortalized human microglia alone or in the presence of SI protein. TNF-alpha was determined 24 hr later by RT-PCR. As can be seen in Figure 8C, unmodified EVs and EVs loaded with GFP behaved similarly and produced a modest, though significant reduction in TNFa. The EVs also expressing CBD produced an even stronger effect, which was significantly superior to the unmodified EVs. These data indicate that the EVs likely work through a distinct pathway from these two therapeutics and allow for a more robust therapeutic regimen for treating viral infections.
  • Example 7 MSCs and/or EVs in the treatment of tissue fibrosis
  • Kidney fibrosis CH-EVs were loaded by electroporation with one of three anti inflammatory molecules: miR-532, miR-190 or anti-miR-214. Human kidney fibroblasts were treated with recombinant SI protein (100 ng/ml) for 24 hr with or without the loaded EVs.
  • the SI protein induced an increase in TGF-b (a marker of cell fibrosis) and unmodified CH-EVs produced a modest, though significant, reduction in this increase.
  • EVs loaded with miR-532, miR-190 or an anti-miR-214 produced an even stronger inhibition on the fibrotic effect of SI.
  • the unmodified MSCs also produced a reduction in TGF-b which was similarly enhanced with the CH-MSCs was loaded with miR-190 (Fig. 9A).
  • Lung fibrosis CH-EVs were loaded by electroporation with one of three anti-inflammatory molecules: miR-29c, miR-328 or anti-miR-214.
  • Human lung fibroblasts were treated with recombinant SI protein (100 ng/ml) for 24 hr with or without the electroporated CH-EVs.
  • SI protein 100 ng/ml
  • EVs loaded with miR-29c, miR-328 or an anti-miR-214 significantly inhibited the effect of SI.
  • CH-MSCs loaded with miR-190 also inhibited the expression of TGF-beta.
  • Lung fibrosis and inflammation- CH or cord MSCs or EVs carrying anti-miR-214, anti- miR-181, anti-miR-214, anti-miR-1246, anti-miR-199, or overexpressing miR-29c, miR-27a, miR-31, miR-124, miR-127 or miR-26a were highly effective.
  • Cystic fibrosis - MSCs or EVs with anti-fibrotic effects can be used for the treatment of cystic fibrosis.
  • This MSCs or EVs can be loaded with the antisense oligonucleotide Eluforsen.
  • MSCs or MSC-derived exosomes carrying miR-29, and/or anti-miR-214 and/or Eluforsen are particularly effective.
  • Liver fibrosis - MSCs with agents that silence TRIB3 (mediates autophagy impairment) or their EVs are particularly effective. This silencing can be done using antisense or siRNA, miR- 30a, miR-9, and/or miR-92-3p.
  • MSCs or EVs carrying the following miRNAs: miR-30d, miR-140p, miR-532 or miR-190 are highly effective.
  • Combinations of miRNA mimics or pre-miRNAs with anti-miRs is also highly effective.
  • This Example shows the tissue fibrosis, including but not limited to severe hyperinflammatory responses and in chronic pathological conditions following severe hyperinflammatory responses (e.g., ARDS, and cytokine storm) and in chronic pathological conditions, can be treated using subpopulations of MSCs and EVs.
  • severe hyperinflammatory responses e.g., ARDS, and cytokine storm
  • chronic pathological conditions e.g., ARDS, and cytokine storm
  • Example 8 MSCs and/or EVs for inhibiting a viral protein priming protein
  • TMPRSS2 is known to function in combination with ACE2 to facilitate SARS-Cov-2 entry into cells.
  • EVs isolated from CH-MSCs or bovine milk were loaded with various miRNA mimics by electroporation. These miRs were hypothesized to decrease TMPRSS2 expression and thus would enhance the anti-viral entry effect of the EVs. To test this, the EVs were incubated with HEK293 cells overexpressing a 3’-UTR of TMPRSS2 luciferase reporter plasmid.
  • EVs regardless of whether they were from CH-MSCs or milk, that expressed let-7, miR-98-5p or miR- 4458 were found to significantly inhibit luciferase expression, indicating that they do indeed target TMPRSS2 (Fig. 10). miR-504 expressing EVs (negative control) had no effect on luciferase expression.

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Abstract

La présente invention fournit des cellules souches mésenchymateuses (MSC) et des vésicules extracellulaires comprenant des protéines incorporées dans une membrane exogène. Des compositions pharmaceutiques comprenant des CSM et des vésicules extracellulaires sont également fournies. La présente invention fournit en outre une méthode de traitement, de prévention ou d'amélioration d'une infection virale, d'une inflammation et/ou d'une fibrose tissulaire.
PCT/IL2021/050274 2020-03-12 2021-03-11 Cellules stromales mésenchymateuses et vésicules extracellulaires pour le traitement d'infections virales, d'inflammation et de fibrose tissulaire WO2021181399A1 (fr)

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US17/920,858 US20230183809A1 (en) 2020-04-22 2021-04-22 Extracellular vesicles for treatment and diagnosis

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